Human sperm protein encyclopedia and alloantigen index: mining novel allo-antigens using sera from ASA-positive infertile patients and vasectomized men.

Anti-sperm antibodies (ASA) are an important cause of immunological infertility. The objective of this study was to identify immunodominant sperm antigens recognized by anti-sperm antibodies (ASA) in serum samples of infertile men, women and vasectomized men. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing (IEF) or nonequilibrium pH gradient electrophoresis (NEPHGE), followed by PAGE and Western blotting. Serum samples from five infertile male and five infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT), were analyzed by Western blotting using NEPHGE gels followed by enhanced chemiluminescence (ECL) to identify the basic sperm antigens reactive to the sera. Serum samples from five fertile male and five fertile female subjects that were ASA-negative by IBT were used as controls. Serum samples from six vasectomized men collected before vasectomy and at different time intervals until 6 months after vasectomy were analyzed by Western blotting using IEF gels. The ECL blots were analyzed to compare immunoreactivity between serum samples from fertile and infertile subjects and identify antigens unique to sera of the infertile subjects. Similarly, immunoreactivity between serum samples from pre- and post-vasectomy was compared to identify antigens unique to sera collected following vasectomy. Five allo-antigenic basic protein spots were recognized by sera from infertile males but not from fertile subjects. Five sperm iso-antigenic basic spots were recognized by infertile female subjects. Two among six of the vasectomized men's sera showed a difference in the Western blot profile 6 months after vasectomy, recognizing at least one new protein spot in each case when compared to pre-vasectomy sera. The acrosomal protein SP-10 was identified as an alloantigen recognized by a post-vasectomy serum. Molecular identities of the known allo- and iso-antigens identified in this study and in previous studies from this laboratory are reviewed and discussed.

Ability of abnormally-shaped human spermatozoa to adhere to and penetrate zona-free hamster eggs: correlation with sperm morphology and postincubation motility.

A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 x 10(6) motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that assessment of morphology may be an unreliable measure, for the individual, of sperm fertilizing ability and emphasize that sperm function testing is an important part of the evaluation of teratospermia.

The role of multifunctional adhesion molecules in spermatogenesis and sperm function: Lessons from hemostasis and defense?

The functional maintenance of the vascular endothelial cell barrier depends on different homo- and heterotypic adhesion systems involving tight junctions, junctional adhesion proteins, and cadherins. Upon inflammatory responses, vessel wall-dependent adhesion and transmigration of leukocytes involves the subtle orchestration of intercellular adhesion receptors and their counter-ligands on each cell type. Following tissue injury, the hemostatic/wound-healing process relies on various cell-associated adhesion receptors (particularly integrins) on platelets and vascular cells as well as on extracellular matrix (ECM) proteins to warrant sealing of the wound. In particular, integrin-binding ECM adhesion molecules mediate firm anchorage as well as cellular motility in cooperation with pericellular proteolytic systems. Accumulating evidence indicates that such cell-anchored and ECM adhesion proteins, which are crucial in vascular defense processes, are also expressed in the testicular epithelium and in gametes to mediate the timely events of spermatid movement during spermatogenesis in the testis and to contribute to the various phases of the fertilization process, culminating in sperm-oocyte fusion, respectively. We explore the multifunctional roles of junctional adhesion molecules, nectins, integrins, ECM proteins, and others beyond their role in defense and hemostasis as important contributors in spermatogenesis and sperm function.

Loss of nectin-2 at Sertoli-spermatid junctions leads to male infertility and correlates with severe spermatozoan head and midpiece malformation, impaired binding to the zona pellucida, and oocyte penetration.

The members of the nectin/CD155 gene family represent a growing class of novel cell adhesion molecules of the immunoglobulin superfamily. In the present study, we describe the generation of a mouse line lacking a functional nectin-2 gene (nectin-2LacZ/LacZ) and analyze the resulting male-specific infertility phenotype. Although nectin-2LacZ/LacZ males produced normal amounts of motile spermatozoa, scanning electron microscopy revealed severe malformations of the spermatozoan head and midpiece. Besides a 4-fold reduction in migration of nectin-2LacZ/LacZ spermatozoa to the oviducts, in vitro binding to zona-intact mouse oocytes was reduced 6-fold. On the other hand, nectin-2LacZ/LacZ spermatozoa bound to zona-free hamster oocytes at near-wild type levels but, remarkably, failed to penetrate. In addition to the previously reported expression of nectin-2 and nectin-3 at Sertoli-spermatid junctions and of nectin-2 at inter-Sertoli cell junctions, we also found nectin-2 to localize at apical cell-cell junctions of the epididymal epithelium. Expression analysis of a LacZ knockin gene into the defunct nectin-2 gene in nectin-2LacZ/LacZ mice provided additional support for our earlier conjecture that in normal testis, nectin-2 is produced exclusively by Sertoli cells. Finally, we found Sertoli-spermatid junctions in nectin-2LacZ/LacZ mice to be virtually devoid of the actin-bundling protein espin, suggesting that ectoplasmic specializations fail to form in the absence of nectin-2. Our functional analyses indicate that the infertility phenotype of nectin-2-deficient male mice is caused by a combination of reduced migration to the oviduct, spermatozoa-zona binding, and sperm-oocyte fusion. We corroborate our previous description of a heterotypic adhesion complex between Sertoli cells and elongated spermatids that is maintained by nectin-2 and nectin-3, respectively.

Surface expression of complement receptor gC1q-R/p33 is increased on the plasma membrane of human spermatozoa after capacitation.

Evidence is increasing that complement components might play a role in fertilization. C1q, the first component of the classical complement cascade, has the ability to promote sperm agglutination in a capacitation-dependent manner as well as an effect on sperm-oolemma binding and fusion. We have previously detected gC1qR, the receptor for the globular head portion of C1q, on the surface of capacitated sperm. In this study, we examined the expression of gC1qR in both fresh and capacitated human spermatozoa. We performed immunoprecipitation for gC1qR and analyzed biotinylated sperm membrane by Western blot to illustrate an increase in receptor density after overnight capacitation. These results were confirmed by flow cytometric analysis of spermatozoa using fluorescein isothiocyanate-labeled monoclonal anti-gC1qR antibody. Confocal, indirect immunofluorescence microscopy revealed an increase in receptor expression over the rostral portion of the sperm head after capacitation. In addition, the ability of live spermatozoa to bind to monoclonal anti-gC1qR antibody-coated microtiter wells was also increased after capacitation. These results suggest that gC1qR may play a role in human fertilization.